1. Field of the Invention
This patent disclosure is concerned with a new fermentation process for the culture of Neisseria meningitidis which incorporates a wholly synthetic medium and event based processing decisions. This process is capable of generating cells at production scale for the extraction and purification of any material which can be produced by culture of Neisseria meningitidis, and specifically, for isolation of an outer membrane protein complex (OMPC) which is useful, among other things, as a protein carrier for conjugate vaccines including PedvaxHIB.RTM. and pneumococcal conjugate vaccines.
2. Background of the Invention
The Outer Membrane Protein Complex (OMPC) of the bacterium Neisseria meningitidis has been used successfully as a protein carrier in a Haemophilus influenza type b (Hib) polysaccharide conjugate vaccine and a Streptococcus pneumoniae (Pn) polysaccharide conjugate vaccine to elicit antipolysaccharide immune responses in young infants. These vaccines are produced by conjugating one, poorly immunogenic component (polysaccharide) with a second, strongly immunogenic component (OMPC). U.S. Pat. No. 4,695,624 described a method for preparing conjugates of poorly immunogenic bacterial polysaccharide and the strongly antigenic Neisseria meningitidis outer membrane protein complex (OMPC). Within the scope of that patent was a method for preparing a commercially available product, PedvaxHIB.RTM., which is a conjugate of OMPC and the capsular polysaccharide of Haemophilus influenzae type b.
Various methods are known for purifying OMPC from gram negative bacteria [Frasch et al., J, Exp. Med. 140, 87 (1974); Frasch et al., J. Exp, Med. 147, 629 (1978); Zollinger et al., U.S. Pat. No. 4,707,543 (1987); Helting et al., Acta Path. Microbio. Scand. Sect. C 89, 69 (1981 ); Helting et al., U.S. Pat. No. 4,271,147; Frasch et al., U.S. Pat. No. 4,601,903; and Vandevelde et al., U.S. Pat. No. 4,451,446]. One method for preparing OMPC, described by Helting et al., in U.S. Pat. No. 4,271,147, comprised detergent extraction of the OMPC of Neisseria meningitidis cells grown in culture, followed by purification of the OMPC. According to the Helting scheme, purification of OMPC is achieved by repeated ultracentrifugation steps and/or ethanol precipitation. Any of these known methods for purification of OMPC are applicable to the Neisseria meningitidis cells produced according to the instant patent disclosure for use in production of conjugate vaccines. However, fermentation methods which provide an inconsistent material from which the OMPC is to be extracted negatively impacts on all of the foregoing OMPC extraction/purification schemes. An efficient and consistent method for commercial scale fermentation of Neisseria meningitidis for production of OMPC has been lacking.
The OMPC carrier is derived by detergent extraction of whole cells. Thus, the consistent generation of suitable biomass is central to an effective OMPC production process. Existing processes for culturing Neisseria employ complex media for fermentation and time associated criteria for batch termination. Serious faults which negatively impact on product yield and quality occur in the known fermentation processes. These processes are at the mercy of lot to lot variation in the quality of complex components in the feed stocks (for example, in the quality of yeast extract); time based process decisions are prone to error due to the stochastic nature of biological systems. These inconsistencies in processing are exacerbated in combination with variations in complex media.
It is widely accepted that the cGMP (U.S. FDA current good manufacturing practice) manufacture of complex biological therapeutics and vaccines must be controlled at all stages of the production process and that final release testing is not itself sufficient to guarantee quality, safety, and efficacy of the final product (FDA Title 21, Code of Federal Regulations, Parts 210, 211, and 600-680). Process validation ensures that the manufacturing operations perform as intended within established specifications. Implicit in this definition is that any given process is well defined and reproducible. Conventional Neisseria fermentation processes rely on chemically undefined or complex media components to speed development time and facilitate robust cultivation, sometimes at the expense of a basic understanding of cell metabolism and strict in-process control. Due to the very nature of complex media components, release testing might not discriminate effectively among lots of complex media; for this reason, the ability to consistently procure a proven grade of complex raw material for fermentation is time consuming and may not always be possible.
Several criteria are central to medium development for microbial fermentation as applied to cGMP biologics production. Ideally, the medium should contain only essential components, be easily prepared in a reproducible manner, and support robust high-cell density culture. A chemically defined medium is inherently more reproducible than a complex medium. Furthermore, a chemically defined medium enables discrete analysis of the effect of each component and strict control of medium formulation through identity and purity testing of raw materials. Finally, the fermentation medium should support the cultivation of the microorganism in question to high-cell density to improve volumetric productivity and to generate a final culture whose composition and physiological condition is suitable for downstream processing. To date, a chemically defined medium and fermentation process for reproducible, high-density cultivation of Neisseria has not been available. Catlin [J. Inf. Dis. 128:178-194, 1973], (hereinafter referred to as "Catlin"), who was principally interested in developing a taxonomy of Neisseria strains based on nutritional requirements rather than large scale fermentation, described a complex chemically defined medium named NEDF, containing approximately 54 ingredients, including all twenty naturally occurring amino acids, for growth of Neisseria. In addition, Catlin described a medium called MCDA containing 18 ingredients (in mM: NaCl, 100; KCL, 2.5; NH.sub.4 Cl, 7.5; Na.sub.2 HPO.sub.4, 7.5; KH.sub.2 PO.sub.4, 1.25; Na.sub.3 C.sub.6 H.sub.5 O.sub.7.2H.sub.2 O, 2.2; MgSO.sub.4.7H.sub.2 O, 2.5; MnSO.sub.4.H.sub.2 O, 0.0075; L-glutamic acid, 8.0; L-arginine.HCl, 0.5; glycine, 2.0; L-serine, 0.2; L-cysteine HCl.H.sub.2 O, 0.06; sodium lactate, 6.25 mg of 60% syrup/mLof medium; glycerin, 0.5% (v/v); washed purified agar, 1% (wt/vol) CaCl.sub.2.2H.sub.2 O, 0.25; Fe.sub.2 (SO.sub.4).sub.3, 0.01 ) which was reported to support growth of Neisseria meningitidis on agar. The ability of MCDA to support growth in liquid medium (that is absent addition of agar) was not reported.
La Scolea et al., [Applied Microbiology 28:70-76, 1974] who like Catlin (see above) was also interested in defining the nutritional requirements of Neisseria strains, reported on the development of a defined minimal medium named GGM for the growth of Neisseria gonorrhoeae. The medium contained minimal salts, eight amino acids, two nitrogen bases, vitamins, coenzymes, metabolic intermediates and miscellaneous components. La Scolea et al., reported growth of this strain to an optical density of 400 Klett units. An absorbance of 1 at 600 nm is considered equivalent to 500 Klett units [see Gerhardt et al., Manual of Methods for General Bacteriology, 1981, ASM., p. 197]. Therefore, the maximum reported growth density achieved by LaScolea et al., was less than about one (1) absorbance unit.
SU 1750689 A1 described a method for preparing polysaccharide-protein vaccines against Neisseria meningitidis B. A defined medium was described having the following composition, g/L:
______________________________________ Sodium L-glutamate 1.30 .+-. 0.10 L-cysteine hydrochloride 0.03 .+-. 0.01 Potassium chloride 0.09 .+-. 0.01 Sodium chloride 6.00 .+-. 1.00 Magnesium sulfate heptahydrate 0.06 .+-. 0.01 Ammonium chloride 1.25 .+-. 0.01 Disubstituted sodium phosphate 2.50 .+-. 0.20 dodecahydrate Trisubstituted sodium citrate 0.50 .+-. 0.10 Glucose 1.60 .+-. 0.20 ______________________________________
In this medium, it is reported that Neisseria may be cultured to a final optical density of 1.5.+-.0.2 on the FEK-56M scale. This is an unfamiliar scale for optical density determination. However, based on the available carbon sources in the above noted medium, it is predictable that the maximum absorbance achievable would be in the range of about 1.5 absorbance units.
In this patent disclosure, we describe a large-scale, high-cell density (5 g/L dry cell weight, and an optical density of between about 10-13 at 600 nm) fermentation process for the cultivation of N. meningitidis. Since current requirements for the production of human biologics mandate strict control of all aspects of the manufacturing process, several key features of the process, including a chemically defined medium and a rational, event-based harvest criterion, support current good manufacturing practice (cGMP) and increased productivity.